Heterotrimeric guanine nucleotide-binding proteins (G-proteins), which consist of ?, , and ? subunits, are bound to hepta-helical serpentine transmembrane G-protein coupled receptors (GPCRs) and regulate intracellular signaling cascades in response to GPCR activation. The ? subunit contains the guanine nucleotide (GDP/GTP) binding site. Upon activation of a GPCR by a neurotransmitter or a hormone, an inactive (GDP-bound) G-protein exchanges GDP for GTP in the ? subunit. The binding of GTP induces a conformational change in the ? subunit and promotes dissociation of the ? subunit from the ? complex. The newly active (GTP-bound) ? subunit and the ? complex interact with downstream effectors. Hydrolysis of GTP terminates activation and returns the G-protein back to its inactive conformation. In this investigation, we will use a battery of spectroscopic an computational methods to characterize the folding of Gi?1 and Gs?, which are the ? subunits that, respectively, inactivate or activate the effector adenylyl cyclase (AC), and thereby decrease or increase the production of the second messenger cAMP. Point mutations in Gi?1 or Gs? at the interface with AC have been implicated in numerous tumors found in the human body. In many disease states, protein aggregates rich in -sheet are present. Using circular dichroism (CD), we found that, in wild type (WT) G? subunits, an increase in temperature induced conversion of ?-helix into -sheet. We hypothesize that G? proteins with hot spot mutations at the interface with AC have a higher percent content in -sheet than WT G?. The hypothesis-driven specific aims of the project are: 1) To probe the accessibility of buried Trp and of more solvent-exposed Tyr residues during folding; and 2) To determine how the secondary structure and ?-cation interactions between Trp and Arg residues in G? subunits impact folding; and 3) to ascertain how interactions between AC and Gi?1 or Gs? mutants differ from those with WT proteins. The methods that will be used to accomplish these goals include, in addition to CD, tryptophan fluorescence, UV detection of tyrosine, isothermal titration calorimetry, nuclear magnetic resonance, X-ray crystallography and molecular dynamics. Besides furthering the understanding of the involvement of G? mutants in cancer, the proposed investigation will address fundamental questions in the fields of protein folding, signal transduction and metallobiochemistry. The AREA project will introduce to research two Ph.D. graduate students, one African-American and the other Hispanic, and, at a minimum, two undergraduate students per year. Additional undergraduates who will conduct research in our laboratory will be supported by University-sponsored research fellowships. The award would therefore increase the research opportunities in biophysical chemistry for students at the Department of Chemistry and Biochemistry and, in turn, at the Lake Side campus of Loyola University Chicago.